In the new era of genomics, proteomics and bio-informatics, a vast number of proteins, new drug targets and small molecules are being investigated intensively and in high-throughput fashion. Although the full mapping of the human genome is done, genomics cannot provide a complete understanding of cellular processes which involve functional interactions between proteins and other molecules as well. Therefore, proteomics may be considered as a cutting-edge area of research today, bridging genomics and cell function.
Current technological methods for analyzing a large number of functional interactions between bio-molecules (especially proteins) include well-plate based screening systems (e.g., ELISA), cell-based assays, soluble reactants screening (e.g., radio immunoassays) and solid-phase assays (e.g., DNA-chips). Today, there is an obvious lack of high throughput technology which enables real-time, label-free monitoring of kinetics of multiple bio-molecular interactions (especially proteins).
The major current limitation in developing such solid-phase based-assays stems from the complexity and variability of proteins. Proteins, in contrast to DNA molecules which are used in producing DNA-chips, are less stable, and generally must kept hydrated and in an active structure and conformation. Also, proteins are very sensitive to chemical and physical changes (e.g., temperature). Finally, with regard to solid-phase kinetic studies, the amount or capacity of an immobilized protein must be known in order to perform an accurate, full kinetic study.
As used herein, the term “biosensor” refers to combination of a receptor surface for molecular recognition and a transducer for generating signals indicative of binding to the surface.
Various related optical methods can be used to measure kinetic binding interactions between bio-molecules. These include, among others, Surface Plasmon Resonance (SPR), total internal reflection fluorescence (TIRF) and evanescent wave elipsometry. It is known in the art to use biosensors and mainly SPR for such purpose. A kinetic binding reaction involves a first molecular species referred to herein as “the probe”. The probe is adsorbed to the sensor surface, and a solution containing a second molecular species, referred to herein as “the target” is then allowed to flow over the probe molecules adsorbed onto the sensor surface. As is known in the art and in commercially available devices, a standard kinetic binding interaction measurement can be described by the following procedure:                (1) Chemical activation of solid-phase surface with a chemical activator (e.g., EDC/NHS); (2) Immobilization of a ‘probe’ molecule on a chemically-activated surface; (3) Washing and blocking of un-occupied activated groups with a blocker such as 1M ethanolamine; (4) Addition of one concentration of a ‘target’ molecule; (5) Washing and regeneration of the ‘probe’ with appropriate regenerating chemicals (e.g., 50 mM NaOH, 0.05% SDS); (6) Addition of another concentration of ‘target’; (7) Repeat stages 4-6, at least five times, each time with a different ‘target’ concentration.        
In one aspect of this invention, the invention provides a method, referred to herein as “One-Shot Kinetics” (OSK). for obtaining one or more kinetic parameters of a binding reaction As shown below, this method allows carrying out a plurality of binding reactions without the need of the regeneration stage which is known to be harmful to the ‘probe’.
In general, any binding event between probe and target molecules can alter an SPR detection parameter which is than is used to monitor the binding reaction. The change in the detection parameter over time is used to determine a characteristic of the binding reaction, such as an association or dissociation constant rates as well as affinity. It is known to use surface Plasmon resonance (SPR) as the method of detection. SPR devices and methods are very sensitive to changes in an optical property of a probe layer and have proven to be useful in detecting changes in an optical property of a probe layer generated by relatively small stimuli.
An SPR probe layer may be configured as a multi-analyte “microarray” in which at each of a plurality of discrete regions, “microspots” on the sensor surface a probe material for interaction with a target material is adsorbed. Berger et al., describes a method for preparing a probe array and for presenting targets to the probe array so as to monitor the binding of the targets to the probes (“Surface Plasmon Resonance Multi-sensing”, Anal. Chem. Vol. 70, February 1998, pp 703-706).
PCT publication WO 02/055993, discloses the use of electrostatic fields and chemical cross-linking for binding probes to a sensor surface.